Regulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as a crucial regulator of long-range chromatin interactions. CTCF often controls specific interactions by preventing inappropriate communication between neighboring regulatory elements or independent chromatin domains. Although recent gene targeting experiments demonstrated that the presence of the CTCF protein is not required for the process of V(D)J recombination per se, CTCF turned out to be essential to control order, lineage specificity and to balance the Ig V gene repertoire. Moreover, CTCF was shown to restrict activity of κ enhancer elements to the Ig κ locus. In this review, we discuss CTCF function in the regulation of V(D)J recombination on the basis of established knowledge on CTCF-mediated chromatin loop domains in various other loci, including the imprinted H19-Igf2 locus as well as the complex β-globin, MHC class II and IFN-γ loci. Moreover, we discuss that loss of CTCF-mediated restriction of enhancer activity may well contribute to oncogenic activation, when in chromosomal translocations Ig enhancer elements and oncogenes appear in a novel genomic context.

Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT-PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated.

Human and mouse studies performed over the last decade have established that follicular helper T (Tfh) cells are a CD4(+) helper subset specialized in the provision of help to B cells. Tfh differentiation is driven by expression of the transcriptional repressor B-cell lymphoma-6 (Bcl-6), which turns on a program that guides T cells close to B-cell areas where Tfh cells first provide help to B cells. Sustained Bcl-6 expression promotes the entry of Tfh cells into follicles and modulates their cytokine expression profile so they can support and select germinal center B cells that have acquired affinity-enhancing mutations in their immunoglobulin genes. Forkhead box 3 protein (Foxp3)(+) regulatory T cells and invariant natural killer T (NKT) cells can also co-opt the Bcl-6-dependent follicular differentiation pathway to migrate into B-cell follicles and regulate antibody responses. The resulting NKT follicular helper cells drive a distinctive type of T-dependent B-cell response to lipid-containing antigens, whereas FoxP3(+) follicular regulatory (Tfr) cells exert a suppressive function on germinal centers. Elucidating how Tfr cells are functionally and numerically regulated and the factors that control the balance between Tfh and Tfr cells is likely to be critical for improved understanding of the pathogenesis and progression of autoimmunity and lymphomas of germinal center origin, and generation of effective vaccines.

Maternal deletion of the NESP55 differentially methylated region (DMR) (delNESP55/ASdel3-4(m), delNAS(m)) from the GNAS locus in humans causes autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib(delNASm)), a disorder of proximal tubular parathyroid hormone (PTH) resistance associated with loss of maternal GNAS methylation imprints. Mice carrying a similar, maternally inherited deletion of the Nesp55 DMR (ΔNesp55(m)) replicate these Gnas epigenetic abnormalities and show evidence for PTH resistance, yet these mice demonstrate 100% mortality during the early postnatal period. We investigated whether the loss of extralarge αs (XLαs) imprinting and the resultant biallelic expression of XLαs are responsible for the early postnatal lethality in ΔNesp55(m) mice. First, we found that ΔNesp55(m) mice are hypoglycemic and have reduced stomach-to-body weight ratio. We then generated mice having the same epigenetic abnormalities as the ΔNesp55(m) mice but with normalized XLαs expression due to the paternal disruption of the exon giving rise to this Gnas product. These mice (ΔNesp55(m)/Gnasxl(m+/p-)) showed nearly 100% survival up to postnatal day 10, and a substantial number of them lived to adulthood. The hypoglycemia and reduced stomach-to-body weight ratio observed in 2-d-old ΔNesp55(m) mice were rescued in the ΔNesp55(m)/Gnasxl(m+/p-) mice. Surviving double-mutant animals had significantly reduced Gαs mRNA levels and showed hypocalcemia, hyperphosphatemia, and elevated PTH levels, thus providing a viable model of human AD-PHP-Ib. Our findings show that the hypoglycemia and early postnatal lethality caused by the maternal deletion of the Nesp55 DMR result from biallelic XLαs expression. The double-mutant mice will help elucidate the pathophysiological mechanisms underlying AD-PHP-Ib.

Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.

Pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase Cδ (PKCδ), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-κB and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in the generation of PKA C-α; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

The potential of antibodies raised against insulin-like growth factor-1 (IGF-1) as a treatment to enhance the anabolic actions of IGF-1 has been demonstrated in both rodent and ruminant models. We investigated whether treatment of genetically normal rats with anti-IGF-1 immunoglobulin (Ig, raised in cattle) would enhance growth and if anti-IGF-1 Ig treatment would ameliorate live-weight loss in genetically normal rats offered a severely protein-restricted diet. Scatchard analysis was used to characterise ammonium sulphate precipitated bovine anti-IGF-1 Ig. Anti-IGF-1 Ig binding to 125I-IGF-1 yielded an almost linear Scatchard plot, with a Hill co-efficient of 0.951 ± 0.012, indicating a single class of IGF-1 binding sites. The affinity of anti-IGF-1 Ig for IGF-1 was 2.14 ± 0.66 × 109 l/mol. The non-immune Ig preparation did not bind IGF-1. Rats were offered either a diet with a normal protein level (20%) or protein restricted (4% protein), and each dietary group was further treated with twice-daily i.p. injections of either diluent phosphate buffered saline, non-immune Ig or anti-IGF-1 Ig. Dietary protein level had a significant effect on live-weight gain, but there was no effect of non-immune Ig or anti-IGF-1 Ig on live-weight gain. Treatment with anti-IGF-1 Ig prevented the significant depression of cumulative dietary intake observed in diluent, and non-immune Ig treated groups offered the 4% protein diet. The cumulative dietary intake of the anti-IGF-1 Ig treated, 4% dietary protein group did not differ significantly from those of the groups offered the 20% protein diet. In addition, within the 4% dietary protein group, rats treated with non-immune Ig exhibited a cumulative feed intake that was intermediate between that of the diluent treated and anti-IGF-1 Ig treated groups (P < 0.05). Size exclusion chromatography was used to characterise in vitro 125I-IGF-1 binding in end-point plasma from each treatment group. In comparison to control groups, anti-IGF-1 Ig treatment resulted in substantially increased 125I-IGF-1 binding in the 30 to 40 kDa region and a concomitant reduction in elution of free 125I-IGF-1. Protein restriction markedly depressed IGF-1 binding at ∼150 kDa in the plasma of diluent and non-immune Ig treated groups. Anti-IGF-1 Ig treatment was effective in preventing this decrease in ∼150 kDa binding. Thus, anti-IGF-1 Ig appears to have a beneficial effect on dietary intake in protein-restricted rats, which is associated with induced changes in IGF-1 binding profiles in plasma.

A wide range of Ca2+ signalling systems deliver the spatial and temporal Ca2+ signals necessary to control the specific functions of different cell types. Release of Ca2+ by InsP3 (inositol 1,4,5-trisphosphate) plays a central role in many of these signalling systems. Ongoing transcriptional processes maintain the integrity and stability of these cell-specific signalling systems. However, these homoeostatic systems are highly plastic and can undergo a process of phenotypic remodelling, resulting in the Ca2+ signals being set either too high or too low. Such subtle dysregulation of Ca2+ signals have been linked to some of the major diseases in humans such as cardiac disease, schizophrenia, bipolar disorder and Alzheimer's disease.

A blood test may be a more acceptable routine colorectal cancer (CRC) screening test than faecal occult blood test, flexible sigmoidoscopy or colonoscopy, and could be safer and cheaper. We evaluated the accuracy of a serum matrix metalloproteinase (MMP9) test for CRC in a non-presenting symptomatic population.

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity.

The advent of reprogramming and its impact on stem cell biology has renewed interest in lineage restriction in mammalian embryos, the source of embryonic (ES), epiblast (EpiSC), trophoblast (TS), and extraembryonic endoderm (XEN) stem cell lineages. Isolation of specific cell types during stem cell differentiation and reprogramming, and also directly from embryos, is a major technical challenge because few cell-surface proteins are known that can distinguish each cell type. We provide a large-scale proteomic resource of cell-surface proteins for the four embryo-derived stem cell lines. We validated 27 antibodies against lineage-specific cell-surface markers, which enabled investigation of specific cell populations during ES-EpiSC reprogramming and ES-to-XEN differentiation. Identified markers also allowed prospective isolation and characterization of viable lineage progenitors from blastocysts by flow cytometry. These results provide a comprehensive stem cell proteomic resource and enable new approaches to interrogate the mechanisms that regulate cell fate specification.

The molecular chaperone HSP90 maintains the activity and stability of a diverse set of "client" proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art SILAC technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention.

The potential for self-reactive T cells to cause autoimmune disease is held in check by Foxp3(+) regulatory T cells (Tregs), essential mediators of peripheral immunological tolerance. Tregs have the capacity to suppress multiple branches of the immune system, tightly controlling the different subsets of effector T cells across multiple different tissue environments. Recent genetic experiments have found mutations that disrupt specific Treg: effector T cell relationships, leading to the possibility that subsets of Tregs are required to suppress each subset of effector T cells. Here we review the environmental factors and mechanisms that allow Tregs to suppress specific subsets of effector T cells, and find that a parsimonious explanation of the genetic data can be made without invoking Treg subsets. Instead, Tregs show a functional and chemotactic plasticity based on microenvironmental influences that allows the common pool of cells to suppress multiple distinct immune responses.

Abstract Differentiation of extra-embryonic tissues and organs, notably the placenta, is vital for embryonic development and growth throughout gestation, starting from a few days after fertilization when the trophoblast cell lineage arises until parturition. In utero metabolic programming events may even extend the impact of placental function well into adulthood as they may predispose the offspring to common pathologies such as diabetes and cardiovascular disease. This review summarizes key steps that lead up to formation of a functional placenta. It highlights recent insights that have advanced our view of how early trophoblast expansion is achieved and how sufficient maternal blood supply to the developing fetus is secured. Exciting cumulative data have revealed the importance of a close cross-talk between the embryo proper and extra-embryonic trophoblast cells that involves extracellular matrix components in the establishment of a stem cell-like niche and proliferation compartment. Remarkably, placental function also relies on beneficial interactions between trophoblast cells and maternal immune cells at the implantation site. Our growing knowledge of the molecular mechanisms involved in trophoblast differentiation and function will help to devise informed approaches aimed at deciphering how placentation is controlled in humans as an essential process for reproductive success and long-term health.

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.

The development of most autoimmune diseases includes a strong heritable component. This genetic contribution to disease ranges from simple Mendelian inheritance of causative alleles to the complex interactions of multiple weak loci influencing risk. The genetic variants responsible for disease are being discovered through a range of strategies from linkage studies to genome-wide association studies. Despite the rapid advances in genetic analysis, substantial components of the heritable risk remain unexplained, either owing to the contribution of an as-yet unidentified, "hidden," component of risk, or through the underappreciated effects of known risk loci. Surprisingly, despite the variation in genetic control, a great deal of conservation appears in the biological processes influenced by risk alleles, with several key immunological pathways being modified in autoimmune diseases covering a broad spectrum of clinical manifestations. The primary translational potential of this knowledge is in the rational design of new therapeutics to exploit the role of these key pathways in influencing disease. With significant further advances in understanding the genetic risk factors and their biological mechanisms, the possibility of genetically tailored (or "personalized") therapy may be realized.

Among the most intensively studied systems in molecular biology is the eukaryotic transcriptional apparatus, which expresses genes in a regulated manner across hundreds of different cell types. Several studies over the past few years have added weight to the concept that transcription takes place within discrete 'transcription factories' assembled inside the cell nucleus. These studies apply innovative technical approaches to gain insights into the molecular constituents, dynamical behaviour and organizational regulators of transcription factories, providing exciting insights into the spatial dimension of transcriptional control.

Over the past two decades, our understanding of phospoinositide 3-kinases (PI3Ks) has progressed from the identification of an enzymatic activity associated with growth factors, GPCRs and certain oncogene products to a disease target in cancer and inflammation, with PI3K inhibitors currently in clinical trials. Elucidation of PI3K-dependent networks led to the discovery of the phosphoinositide-binding PH, PX and FYVE domains as conduits of intracellular lipid signalling, the determination of the molecular function of the tumour suppressor PTEN and the identification of AKT and mTOR protein kinases as key regulators of cell growth. Here we look back at the main discoveries that shaped the PI3K field.

Lymphocyte development requires cells to progress through a series of stages, each associated with changes in gene expression. Intense effort has been invested into characterising the dynamic networks of transcription factors underlying these regulated changes. Whilst transcription factors determine the tempo at which mRNA is produced, recent results highlight the importance of the selective regulation of mRNA decay and translation in regulating gene expression. These processes are regulated by sequence-specific RNA-binding proteins (RBP) as well as noncoding RNA such as microRNAs. RNA-binding proteins are emerging as important regulators of cell fate and function in both developing and mature lymphocytes. At the molecular level the function of RNA-binding proteins is integrated with signal transduction pathways that also govern gene transcription.

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

In this study, we present an innovative mathematical modeling approach that allows detailed characterization of Ca(2+) movement within the three-dimensional volume of an atrial myocyte. Essential aspects of the model are the geometrically realistic representation of Ca(2+) release sites and physiological Ca(2+) flux parameters, coupled with a computationally inexpensive framework. By translating nonlinear Ca(2+) excitability into threshold dynamics, we avoid the computationally demanding time stepping of the partial differential equations that are often used to model Ca(2+) transport. Our approach successfully reproduces key features of atrial myocyte Ca(2+) signaling observed using confocal imaging. In particular, the model displays the centripetal Ca(2+) waves that occur within atrial myocytes during excitation-contraction coupling, and the effect of positive inotropic stimulation on the spatial profile of the Ca(2+) signals. Beyond this validation of the model, our simulation reveals unexpected observations about the spread of Ca(2+) within an atrial myocyte. In particular, the model describes the movement of Ca(2+) between ryanodine receptor clusters within a specific z disk of an atrial myocyte. Furthermore, we demonstrate that altering the strength of Ca(2+) release, ryanodine receptor refractoriness, the magnitude of initiating stimulus, or the introduction of stochastic Ca(2+) channel activity can cause the nucleation of proarrhythmic traveling Ca(2+) waves. The model provides clinically relevant insights into the initiation and propagation of subcellular Ca(2+) signals that are currently beyond the scope of imaging technology.